The research proposed in this application deals with the control of urinary acidification in the collecting tubule (CT). We propose that this process is controlled by two pumps, an H-ATPase and an H-K-ATPase. We will characterize these pumps, both functionally and biochemically. Two hypotheses will be tested. Hypothesis I - Acidification in the CT is mediated by two proton pumps, an H-ATPase and an H-K-ATPase. The experiments testing this hypothesis will be performed in rat and rabbit nephron fragments. Considerable data concerning the H-ATPase is already available, much less is known about the H-K-ATPase. To show that these enzymes mediate distal acidification it is necessary to show that their activities change appropriately with acid-base perturbations and that their inhibition reduces acidification. We will study inhibition of the H-ATPase with bafilomycin A and N-ethylmaleimide; H-K-ATPase activity will be studied using vanadate, SCH28080, or K removal. The roles of aldosterone, K, and Cl in modulating enzyme activity will be examined. Hypothesis II - Inhibition of the H-K-ATPase results in hypokalemic distal renal tubular acidosis. These studies will be performed in rats and will employ clearance techniques, measurement of enzyme activities along the nephron, and functional studies in isolated tubules. We will study the effects of inhibiting the H-K-ATPase with vanadate and SCH28080. New data show, as predicted, that vanadate induces hypokalemic distal renal tubular acidosis. Other models of distal renal tubular acidosis will also be studied. By studying alterations in whole animal acid-base status and acid excretion, enzyme activities, net acidification in isolated tubules, and cell pH recovery when basolateral proton extension is blocked, we hope to elucidate the relationship between ATPase activity and tubular function.